RNA helicases to combat RNA phase transitions in repeat expansion disorders

RNA解旋酶对抗重复扩增障碍中的RNA相变

• 批准号: 10640592
• 负责人: Linamarie Miller
• 金额: $ 6.87万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10640592
• 关键词: Address; Affect; Affinity; Age of Onset; Antisense Oligonucleotides; Binding; Biochemical; Biological Assay; CAG repeat; CRISPR/Cas technology; Catalogs; Cell Nucleus; Cells; Central Nervous System; Central Nervous System Diseases; Chemicals; Clinical Trials; Code; Collaborations; Collection; Cytoplasm; Cytoplasmic Granules; Disease; Event; Failure; Fellowship; Future; General Population; Genetic; Genome; Human; Huntington Disease; In Vitro; Knock-out; Language; Length; Liquid substance; Mass Spectrum Analysis; Mediating; Methods; MicroRNAs; Molecular; Monitor; Neurodegenerative Disorders; Nuclear; Nuclear RNA; Nucleotides; Pathologic; Pathology; Pediatric Hospitals; Pennsylvania; Peptides; Phase; Phase Transition; Philadelphia; Physical condensation; Proteins; Protocols documentation; RNA; RNA Helicase; RNA Splicing; RNA metabolism; RNA purification; RNA-Binding Proteins; Recombinants; Regulation; Research Institute; Research Personnel; Ribonucleoproteins; Role; Severity of illness; Spinocerebellar Ataxias; Techniques; Therapeutic; Toxic effect; Training; Transcript; Translations; Universities; Untranslated RNA; Visuospatial; Work; candidate identification; cellular targeting; combat; design; executive function; helicase; in vivo; information processing; insight; multimodality; mutant; nervous system disorder; neurotoxic; new therapeutic target; novel therapeutics; physical property; processing speed; protein function; spinal and bulbar muscular atrophy; stress granule; tool; transcriptome sequencing

Abstract Repeat-expansion disorders are a collection of more than 40 rare but devastating diseases that largely effect the central nervous system. Currently, they are incurable. The genetic basis for these disorders is repeat expansions, the length of which determines disease severity and age of onset. In these disorders, repeat sequences cause pathology in a multimodal fashion. The translation of repeats located in coding regions can disrupt normal protein function and produce toxic repeat peptides. These neurotoxic peptides can be produced even when the repeats are in noncoding regions via repeat associated non-AUG translation. Another agent of pathology is the repeat RNA transcript itself, where aberrant binding of the repeat RNA to RNA-binding proteins leads to issues like deregulation of the miRNA machinery, aberrant translation, and mis-splicing events. Repeat RNAs also enable transcripts to undergo liquid-liquid phase separation (LLPS) and form nuclear RNA foci in cells. These foci can sequester RNA-binding proteins, which may contribute to the observed repeat RNA toxicity. It has been well established that RNA helicases regulate the formation of phase separated ribonucleoprotein (RNP) granules in cells, but no work exists to understand how helicases affect the RNA foci in repeat-expansion disorders. Considering first that repeat RNAs cause potentially pathological RNA foci and that second, helicases are important for the formation of other cellular RNP granules, we hypothesize that RNA helicases can also modulate the formation of RNA foci in repeat-expansion disorders. The proposed work is focused on the deleterious RNA foci that form in the repeat-expansion disorders caused by CAG trinucleotide expansions like Huntington’s Disease and several spinocerebellar ataxias. This proposed study aims to identify RNA helicases that modulate these deleterious RNA foci and understand the mechanism by which they do so. Knockout screens in human HAP1 cells expressing CAG repeat constructs which form RNA foci have identified candidate RNA helicases that affect RNA foci size and number. This work will utilize in vitro approaches to understand the mechanism underlying how these candidate helicases effect RNA foci. After the recent failure of two candidate therapies for Huntington’s Disease in clinical trials, it is especially necessary to consider new therapeutic targets for these disorders. This work will expand our understanding of RNA helicase function in repeat-expansion disorders and offer a new therapeutic tool to explore for combatting these diseases. The proposed fellowship will be conducted at the University of Pennsylvania in the lab of Dr. James Shorter, a leading researcher in the molecular basis of neurodegenerative diseases and protein disaggregases. Completion of the proposal in this lab will provide rigorous training in biochemical assay design and in vitro protein and RNA handling techniques.

摘要 重复扩展障碍是40多种罕见但毁灭性疾病的集合， 中枢神经系统目前，他们是无法治愈的。这些疾病的遗传基础是重复 扩张，其长度决定疾病的严重程度和发病年龄。在这些疾病中，重复 序列以多模式方式引起病理。位于编码区的重复序列的翻译可以 破坏正常蛋白质功能并产生有毒重复肽。这些神经毒性肽可以在 即使当重复序列通过重复序列相关的非AUG翻译位于非编码区时。之另一名代理 病理是重复RNA转录本本身，其中重复RNA与RNA结合蛋白的异常结合 导致了诸如miRNA机制失调、异常翻译和错误剪接事件等问题。重复 RNA还使转录物能够经历液-液相分离（LLPS）并在细胞核中形成核RNA灶。 细胞这些病灶可以隔离RNA结合蛋白，这可能有助于观察到的重复RNA毒性。 RNA解旋酶调节相分离的核糖核蛋白的形成 (RNP)颗粒，但没有工作存在，以了解如何解旋酶影响RNA焦点在重复扩增 紊乱首先考虑重复RNA引起潜在的病理性RNA病灶，其次考虑解旋酶， 对于其他细胞RNP颗粒的形成是重要的，我们假设RNA解旋酶也可以 调节重复扩增疾病中RNA灶的形成。拟议工作的重点是 在CAG三核苷酸扩增引起的重复扩增障碍中形成的有害RNA灶， 亨廷顿病和几种脊髓小脑共济失调。本研究旨在鉴定RNA解旋酶 来调节这些有害的RNA灶，并了解它们这样做的机制。淘汰筛 在表达CAG重复构建体的人HAP 1细胞中，所述CAG重复构建体形成RNA灶， 影响RNA焦点大小和数量的解旋酶。这项工作将利用体外方法来了解 这些候选解旋酶如何影响RNA焦点的潜在机制。在最近两名候选人失败后， 在临床试验中，尤其需要考虑新的治疗靶点 治疗这些疾病这项工作将扩大我们对RNA解旋酶在重复扩增中功能的理解 并提供了一种新的治疗工具，以探索对抗这些疾病。 拟议的奖学金将在宾夕法尼亚大学的James Shorter博士的实验室进行， 神经退行性疾病和蛋白质分解的分子基础的主要研究人员。完成 该实验室的建议将提供严格的生化分析设计和体外蛋白质和RNA的培训 处理技术。